Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases

Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface- bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.     

Structural basis of the endoproteinase-protein inhibitor interaction

Proteolytic enzymes are potentially hazardous to their protein environment, so that their activity must be carefully controlled. Living organisms use protein inhibitors as a major tool to regulate the proteolytic activity of proteinases. Most of the inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with the active sites in a 'canonical' i.e. substrate-like manner via an exposed reactive site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors directed against coagulation factors, in particular thrombin, a few cysteine proteinase inhibitors inhibitory towards papain-like proteinases, and three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are presented and briefly discussed with respect to the different strategies applied by nature.     

Leukocyte cell surface proteinases: regulation of expression, functions, and mechanisms of surface localization

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: (1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinases by cells; (2) the availability of surface binding sites for proteinases; and/or (3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: (1) concentrating the activity of proteinases to the immediate pericellular environment; (2) facilitating pro-enzyme activation; (3) increasing proteinase stability and retention in the extracellular space; (4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and (5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes.     

Fluorescence methods for localizing proteinases and proteinase inhibitors in skeletal muscle

Summary Proteinases and proteinase inhibitors have become suspect in a wide variety of muscle wasting conditions that might be treatable if knowledge of the cellular locale and function of these molecules were known. Fluorescent probes have been useful in the localization of proteinases in muscle samples from human and animal specimens. These include the histochemical localization of proteinases based on the specific fluorescence of hydrolysis product derivatives, but this approach has been limited to the lysosomal proteinases because of the acidic requirements of the trapping reaction of the primary reaction product. Immunohistochemical techniques do not have the same restrictions and a number of lysosomal and nonlysosomal proteinases have been identified in muscle by this means. Unfortunately, they do not yield any information as to the activity of the enzymes. This is an important consideration since the extracellular environment contains a number of proteinase inhibitors, some of which may be internalized by the cell.     

Novel roles of protease inhibitors in infection and inflammation

The local balance between proteinase inhibitors and proteinases determines local proteolytic activity. Various studies have demonstrated the importance of serine proteinase inhibitors in regulating the activity of serine proteinases that are released by leucocytes during inflammation. Recently it has been shown that these inhibitors may also display functions that are distinct from those associated with the inhibition of leucocyte-derived proteinases. In this review the results of selected studies focusing on three inhibitors of neutrophil elastase, i.e. alpha(1)-proteinase inhibitor, secretory leucocyte proteinase inhibitor and elafin, are presented, with the aim of illustrating their possible involvement in the regulation of inflammation, host defence against infection, tissue repair and extracellular matrix synthesis.     

Elastase and cathepsin G of human monocytes. Quantification of cellular content, release in response to stimuli, and heterogeneity in elastase-mediated proteolytic activity

Human peripheral blood monocytes contain human leukocyte elastase (HLE) and cathepsin G (CG), serine proteinases originally described in azurophil granules of polymorphonuclear neutrophils (PMN). Immunoreactive HLE and CG of freshly harvested monocytes have been quantified in this study; to begin to elucidate potential roles for these enzymes in extracellular events, release in response to stimuli has been measured, along with proteolytic activity of monocytes toward surface-bound proteins. Our results indicate that whole-cell extracts of monocytes contain approximately 6% of the amount of HLE as do extracts of comparable numbers of PMN. In response to PMA in vitro, monocytes released 39 to 53% of their content of HLE and CG within 60 min, a fractional release greater than that of PMN. Furthermore, when phorbol-stimulated monocytes were adherent to a fibronectin-coated surface, extensive HLE-mediated proteolysis of the surface-bound protein was observed. Proteolysis by such cells in the presence of proteinase inhibitors was of considerable interest, since a subpopulation (15 to 20% of the total) expressed marked but localized proteolytic activity, possibly escaping inhibition through contact-mediated mechanisms. These data indicate that a subpopulation of freshly harvested monocytes is rich in HLE and CG (serine proteinases traditionally associated with PMN), can promptly release HLE and CG in response to stimuli, and can utilize HLE for extracellular proteolysis. Monocyte-derived serine proteinases may participate in extracellular events formerly associated with PMN-derived HLE and CG.     

Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high Kis. In this study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs can be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62% between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0–10.0). When studied by SDS-PAGE in the presence of β-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30–60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0-10.0). When studied by SDS-PAGE in the presence of beta-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30-60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Proteinases involved in matrix turnover during cartilage and bone breakdown

The joint is a discrete unit that consists of cartilage, bone, tendon and ligaments. These tissues are all composed of an extracellular matrix made of collagens, proteoglycans and specialised glycoproteins that are actively synthesised, precisely assembled and subsequently degraded by the resident connective tissue cells. A balance is maintained between matrix synthesis and degradation in healthy adult tissues. Different classes of proteinases play a part in connective tissue turnover in which active proteinases can cleave matrix protein during resorption, although the proteinase that predominates varies between different tissues and diseases. The metalloproteinases are potent enzymes that, once activated, degrade connective tissue and are inhibited by tissue inhibitors of metalloproteinases (TIMPs); the balance between active matrix metalloproteinases and TIMPs determines, in many tissues, the extent of extracellular matrix degradation. The serine proteinases are involved in the initiation of activation cascades and some, such as elastase, can directly degrade the matrix. Cysteine proteinases are responsible for the breakdown of collagen in bone following the removal of the osteoid layer and the attachment of osteoclasts to the exposed bone surface. Various growth factors increase the synthesis of matrix and proteinase inhibitors, whereas cytokines (alone or in combination) can inhibit matrix synthesis and stimulate proteinase production and matrix destruction.     

Extracellular serine proteinase from Bacillus licheniformis

The serine proteinase from B. licheniformis was purified by affinity chromatography on the sorbent obtained by attachment of p-(omega-aminomethyl)-phenylboronic acid via an amino group to CH-Sepharose. The use of this sorbent specific to the serine proteinases active sites resulted in a 35-fold purification of the enzyme with an apparent activity yield of 288%. Such a high activity yield is due to a removal of the enzyme inhibitors. The N-terminal sequence of B. licheniformis extracellular serine proteinase traced for 35 amino acid residues coincides with that of subtilisin Carlberg, a serine proteinase presumed to be secreted by a B. subtilis strain. Since the amino acid composition as well as the functional properties of these two enzymes did not reveal any noticeable differences, it was assumed that both proteinases are very similar, if not identical. This conclusion leads to reconsideration of the existing concept on an extremely fast rate of subtilisin evolution. Three multiple forms of B. licheniformis extracellular serine proteinase were found to differ only in their net charges, presumably as a result of partial deamidation of Asn or Gln residues within their structure.     

Up-regulation of multiple serine proteinases during earthworm tail regeneration

Summary

Previous studies have shown that spatiotemporal regulation of extracellular matrix (ECM) by proteinases is implicated in the initial step of regeneration. In amphibian regeneration, the up-regulation of proteinases such as metalloproteinases (MMPs) and cathepsin D, and proteinase-related proteins such as proteinase tissue inhibitors and activators has been demonstrated. Since the earthworm could provide a unique and valuable model to investigate the mechanism of regeneration, we studied the developmental change in proteinase expression during earthworm tail regeneration. Zymographic analysis revealed that proteinase activities began to increase within 1 h after amputation and reached a maximum at 7 days post-amputation. This peak in activity was approximately 22-fold greater than the unamputated controls. Thereafter, the proteinase activities tended to decrease followed by another peak at 30 days before returning to control levels. At least four types of proteinase were distinguishable at 7 and 30 days post-amputation, with molecular weights of 25, 28, 38, and 44 kDa, respectively. All proteinase activities were strongly inhibited by addition of phenylmethylsulfonyl fluoride (PMSF) and aprotinin, specific inhibitors for serine proteinase. Pepstatin A, E-64, iodoacetamide and a metal ion-free medium were not effective inhibitors, indicating that proteinases expressed during earthworm tail regeneration would be serine proteinases. In addition, we were able to detect two types of plasminogen activator (PA) with molecular weights of 40 and 47 kDa, respectively. PA activities were predominantly expressed at 1, 5, and 25 days post-amputation, which preceded two peaks of serine proteinase activities appearing at approximately 7 and 30 days after amputation, respectively. This fact supports the view that serine proteinases expressed in respond to tail amputation may be plasmin-like proteinases activated by PA.     

Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis

he presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.     

Inhibition of IgA1 proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids

The alpha-aminoboronic acid analog of proline has been synthesized and incorporated into a number of peptides as the COOH-terminal residue. These peptide prolyl boronic acids are potent inhibitors of both the type 1 and type 2 IgA proteinases from Neisseria gonorrhoeae and Hemophilus influenzae, but not of the functionally similar IgA proteinase from Streptococcus sanguis. The best inhibitors synthesized thus far have Ki values in the nanomolar range (4.0 to 60 nM). These results indicate that the N. gonorrhoeae and the H. influenzae enzymes belong to the serine protease family of proteolytic enzymes while that from S. sanguis does not. As a group, the IgA proteinases have been noted for their remarkable specificity; thus, the peptide prolyl boronic acids reported here are the first small synthetic molecules to exhibit a relatively high affinity for the active site of an IgA proteinase and are therefore the first to yield some insight into the active site structure and specificity requirements of these enzymes.     

Antibody raised against extracellular proteinases ofSporothrix schenckii inS. schenkii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin, while proteinase II is an aspartic proteinase, inhibited by pepstatin. Studies on substrate specificity and the effect of proteinase inhibitors on cell growth suggest an important role for these proteinases in terms of fungal invasion and growth. There has, however, been no evidence presented demonstrating thatS. schenckii produces 2 extracellular proteinases in vivo. In order to substantiate the in vivo production of proteinases and to attempt a preliminary serodiagnosis of sporotrichosis, serum antibodies against 2 proteinases were assayed usingS. schenckii inoculated hairless mice. Subsequent to an intracutaneous injection ofS. schenckii to the mouse skin, nodules spontaneously formed and disappeared for a period of 4 weeks. Histopathological examination results were in accordance with the microscopic observations. Micro-organisms disappeared during the fourth week. Serum antibody titers against purified proteinases I and II were measured weekly, using enzyme-linked immunosorbent assay (EIA). As a result, the time course of the antibody titers to both proteinases I and II were parallel to that of macroscopic and microscopic observations in an experimental mouse sporotrichosis model. These results suggest thatS. schenckii produces both proteinases I and II in vivo. Moreover, the detection of antibodies against these proteinases can contribute to a serodiagnosis of sporotrichosis.     

Exoproteinases of the Oomycete Phytophthora infestans

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown (by SDS-PAGE in the presence of gelatin) to include at least six components differing in molecular weight. Inhibitory analysis and studies of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases (specific for trypsin and subtilisin) and metalloproteinases. Their activity is suppressed by proteinase-inhibiting proteins from potato tubers. It is suggested that exoproteinases of P. infestans may be the metabolic target for natural proteinase inhibitors from potato.     

Proteinases and myocardial extracellular matrix turnover

Extracellular structural remodeling is the compensatory response of the tissue following pathological stage. Myocardial infarction, which leads to adverse remodeling, thinning of the ventricle wall, dilatation and heart failure, is one of the leading causes of death. Remodeling implies an alteration in the extracellular matrix and in the spatial orientation of cells and intracellular components. The extracellular matrix is responsible for cardiac cell alignment and myocardial structural integrity. Substances that break down the extracellular matrix, specialized proteinases as well as inhibitors of proteinases, appear to be normally balanced in maintaining the integrity of the myocardium. Myocardial infarction leads to an imbalance in proteinase/ antiproteinase activities causing alterations in the stability and integrity of the extracellular matrix and adverse tissue remodeling. To explore mechanisms involved in this process and, in particular, to focus on matrix metalloproteinases, their inhibitors, and activators, an understanding of proteinase and antiproteinase is needed. This review represents new and significant information regarding the role of activated matrix proteinases antiproteinases in remodeling. Such information will have a significant impact both on the understanding of the basic cell biology of extracellular matrix turnover, as well as on potential avenues for pharmacological approaches to the treatment of ischemic heart disease and failure.     

Cysteine peptidases and their inhibitors in breast and genital cancer

Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.     

Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3

The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.